Studies have shown that supplementation with hyaluronic acid (HA), a glycosaminoglycan found in mammalian follicular, oviduct, and uterine fluids, improves in vitro development and post-thaw survival of bovine embryos. In this study, we examined the effect of HA supplementation on ovine embryo development and on survival after vitrification using the minimum volume cooling (MVC) cryotop method. Abattoir sourced ovine oocytes were in vitro matured and fertilized as per routine procedures (Walker et al. 1996 Biol. Reprod. 55, 703–708). In Experiment 1 (5 replicates), presumptive zygotes were randomly allocated to IVC medium supplemented with 0.8 mg mL–1 BSA, amino acids, and 0, 0.5, 1.0, 1.5 or 2.0 mg mL–1 HA. Cleavage rates were recorded and blastocyst development evaluated on Day 7 (Day 0 = day of IVF). In Experiment 2 (3 replicates), presumptive zygotes were placed in in vitro culture (IVC) medium with or without 1.0 mg mL–1 HA. Embryos were vitrified using the MVC cryotop method (Kelly et al. 2004 Reprod. Fert. Dev. 16, 172) on either Day 5 (morula–blastocyst stages), Day 6 (compact morula–hatching blastocyst stages), or Day 7 (blastocyst–hatching blastocyst stages). Vitrified embryos were thawed 7 days later and placed into IVC medium. Embryo survival (assessed by blastocoele re-expansion) and hatching rates were recorded on Day 8. Variables were assessed using procedure CATMOD in SAS (SAS Institute Inc., Cary, NC, USA). In Experiment 1, the addition of HA did not affect cleavage or blastocyst formation rates but hatching rates were significantly (P < 0.05) improved at concentrations of 0.5 to 1.5 mg mL–1 (Table 1). In Experiment 2, HA supplementation (1.0 mg mL–1) compared with control medium did not affect cleavage (96.8 and 97.1%, respectively) or blastocyst formation rates (68.6 and 70.2%, respectively). HA significantly (P < 0.05) improved survival after thawing of embryos vitrified on Day 5 (100 v. 85.6%, n = 85 and 90). However no effect was observed when embryos were vitrified on either Day 6 (97.8 v. 97.8%, n = 91 and 92) or Day 7 (96.7 v. 97.9%, n = 92 and 94). Within day, HA supplementation did not affect hatching rate compared with control medium (Day 5, 54.1 v. 53.2%; Day 6, 62.9 v. 65.6%; Day 7, 71.9 v. 62.0%, respectively). These results demonstrate that HA supplementation of IVC medium significantly improves hatching rate of ovine embryos and we speculate that this improvement may correlate with comparable improvements in pregnancy rates after transfer. Hyaluronic acid binds to CD44, a glycoprotein expressed on the surface of preimplantation ovine embryos and shown to play a role on embryo development (Luz et al. 2012 Genet. Mol. Res. 11, 799–809). Hyaluronic acid plays a role in cell migration and we suggest that, in the early blastocyst, it affords an advantage to the trophectoderm cells.
Table 1.Effect of hyaluronic acid (HA) supplementation in IVC medium on cleavage, blastocyst, and hatching rates
Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System
